CN1682113A - Fixation carrier and solid phase - Google Patents
Fixation carrier and solid phase Download PDFInfo
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- CN1682113A CN1682113A CNA038216086A CN03821608A CN1682113A CN 1682113 A CN1682113 A CN 1682113A CN A038216086 A CNA038216086 A CN A038216086A CN 03821608 A CN03821608 A CN 03821608A CN 1682113 A CN1682113 A CN 1682113A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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Abstract
The object of the present invention is to provide an immobilization support having an optimum support surface for immobilizing various types of materials having different properties with specificity, efficiency, and good reproducibility, and to provide a solid phase having improved characteristics by use of the above support. The immobilization support of the present invention is provided with an electrolyte thin film on the surface of the support. This immobilization support can be applied to the fabrication of a solid phase used in, for example, an antigen-antibody reaction, nucleic acid hybridization, a receptor assay, or a biosensor.
Description
Technical field
The present invention relates in the antigen-antibody reaction on the solid phase, at the hybridization of the nucleic acid on the solid phase, the fixation support that in the reaction of acceptor on the solid phase and part, enzyme on solid phase and the reaction of matrix, the immobilization of cell etc., uses, and the solid phase of utilizing this carrier.
Background technology
Be used for being determined at antigen-antibody reaction on the solid phase, the hybridization of the nucleic acid on the solid phase, the reaction of acceptor on the solid phase and part, on solid phase the immobilization of the system of the reaction of enzyme and matrix many with carrier be material with bioabsorbable polymer materials such as plastics such as polystyrene and polycarbonate, cellulose, glass, ceramic resin, metal, Markite etc.But in order to control the binding capacity of immobilized material on these carriers, at first consider Material Selection, when passing through to select to seek to make immobilization reach optimization to surface modification under the indeterminable situation of material with carrier by plasma, gamma-radiation, ultraviolet ray or ozone.But optimization method in the past has problems at the aspects such as repeatability of modified condition, and, for solid phases such as microplate and latex particles, be difficult to even modification is carried out on its surface.Especially, only change restricted several condition, for effectively to carry out the best surface modification extremely difficult in conjunction with having diversified material of different nature.For this reason, on solid phase fixedly the Study on Conditions of desired substance need spend a large amount of labours, according to the difference of occasion, also exist can not being completely fixed situation, the immobilized immobilization carrier of material can be freely controlled in therefore urgent hope.
In addition, about the above-mentioned background technology, the patent documentation (the international WO97/19978 volume that discloses) that is entitled as " immobilizing microbial cells with the manufacturing installation of bead-type substrate and bead-type substrate " discloses the immobilizing microbial cells bead-type substrate, the following formation of this carrier: in the water-bearing media that contains alkali metal ion or polyvalent metal ion, drip and contain (a) has at least 2 ethylenic unsaturated links in 1 molecule water wettability light-cured resin, (b) Photoepolymerizationinitiater initiater and (c) are by contacting the fluid composition of the water soluble polymer polysaccharide with gelatinisation ability with alkali metal ion or polyvalent metal ion, it is granular that the said composition gel is turned to, the irradiation active ray makes it to solidify, proportion be 1.00～1.20 and with contact angle that the n-paraffin on surface forms be 2 °～30 °, have the surface that is suitable for attached microbial.But shining this active ray is the intensity (the 14th page the 6th to eighth row) that improves bead-type substrate for the photopolymerization by light-cured resin, in the document, for the further surface modification of gained carrier without any open.
In addition, patent documentation (spy opens the 2000-65832 communique) relates to the pre-treatment of avoiding sample, the filtrator shape biological specificity reaction assay that can access correct testing result is with carrier and use the assay method of this biological specificity reaction assay with carrier, and relate to filtrator shape biological specificity reaction assay carrier, it is characterized in that constituting by the organic polymer of the filtrator shape of fine pore with 1～100 μ m.This filtrator shape organic polymer can further be selected from a kind of processing mode in hydrolysis, corona discharge, Cement Composite Treated by Plasma, UV-ozone treatment, coating carbodiimides (bond) the isoreactivity material and obtain (the 4th page of the 5th hurdle (0011) section and (0015) section) at fiber surface according to prior art at least.
Thereby, the object of the present invention is to provide can be special, the effective and immobilization carrier that repeatability is fixed well, formation optimum carrier surface of the diversified material that makes different in kind, and the solid phase that has the characteristic of improvement by the use of this carrier is provided.
Summary of the invention
Curing of the present invention is characterised in that with carrier, and electrolytic thin-membrane is set in its surface.That is, the 1st aspect of the present invention be,
(1) the immobilization carrier is characterized in that, on the surface of carrier electrolytic thin-membrane is set.
The material of above-mentioned electrolytic thin-membrane also can use inorganic material, preferably uses macromolecular material to form electrolytic thin-membrane.Thereby the 2nd aspect of the present invention be,
(2) the immobilization carrier of above-mentioned (1), described electrolytic thin-membrane is formed by macromolecular material.
Above-mentioned electrolytic thin-membrane can be the film that is made of polyanion or polycation, thereby the 3rd aspect of the present invention be,
(3) the immobilization carrier of above-mentioned (2), described electrolytic thin-membrane is made of polyanion film or polycation film.
Even more preferably, above-mentioned electrolytic thin-membrane can be by with a plurality of different polyanion films or polycation film combination in any and lamination cambium layer press mold.Particularly preferably be, above-mentioned polyanion film and polycation film alternatively laminated form this electrolytic thin-membrane.That is, the 4th aspect of the present invention be,
(4) the immobilization carrier of above-mentioned (2), described electrolytic thin-membrane is formed by polyanion film and polycation film alternatively laminated.
Immobilization of the present invention can be applied in the solid phase of making the hybridization be used for antigen-antibody reaction, nucleic acid, receptor assay, biology sensor etc. with carrier.Particularly, immobilization of the present invention can be used for combining by the material histocyte thalli virus of biosome origin or with them with carrier or have the immobilization of the material etc. of compatibility with them, advantageously, when on stationary phase, supplying the object material of mensuration, can be provided on this solid phase carrier in conjunction with the best surface of solid phase carriers of supplying material paired with the object material.Thereby the 5th aspect of the present invention be,
(5) each immobilization carrier of above-mentioned (1)～(4) is used for fixing and detects material that thing combines or the material with compatibility.
As immobilization of the present invention with carrier on immobilized object, can enumerate protein, glycoprotein, peptide, glycopeptide, polysaccharide, nucleic acid, lipid, glycolipid matter etc. and have the material of compatibility by material, cell or the material that combines with them of biosome origin or with them.Thereby the 6th aspect of the present invention be,
(6) each immobilization carrier of above-mentioned (1)～(5), being used for fixing protein, glycoprotein, peptide, glycopeptide, polysaccharide, nucleic acid, lipid, glycolipid matter etc. have the material of compatibility by material, cell or the material that combines with them of biosome origin or with them.
In addition, the present invention also provides the immobilization of using above-mentioned (1)～(4) the record solid phase that offers various determination tests etc. with carrier, in this solid phase, the material that combines with the detection thing or the material, the especially protein that have compatibility with the detection thing, glycoprotein, peptide, glycopeptide, polysaccharide, nucleic acid, lipid, glycolipid matter etc. have high, the effective and repeatability immobilization well of energy quilt of specificitys such as material of compatibility by material, cell or the material that combines with them of biosome origin or with them.Thereby the 7th and the 8th aspect of the present invention be,
(7) each immobilization of above-mentioned (1)～(4) with carrier on, have will with detect material that thing combine or with detect the substance fixed solid phase that thing has compatibility,
(8) each immobilization of above-mentioned (1)～(4) with carrier on, have protein, glycoprotein, peptide, glycopeptide, polysaccharide, nucleic acid, lipid, glycolipid matter etc. had the substance fixed solid phase of compatibility by material, cell or the material that combines with them of biosome origin or with them.
By above-mentioned each constitute, the present invention can provide can be specifically, effectively and repeatability well with the immobilization carrier of the diversified material combination of different in kind, and provide solid phase with excellent specific property.
Embodiment
Immobilization of the present invention is provided with electrolytic thin-membrane with carrier on the surface of its base material.
As the material of immobilization of the present invention with the base material of carrier, can use and well known to a person skilled in the art any material, can enumerate boiomacromolecule material, glass, ceramic resin, metal, Markites etc. such as plastics such as polystyrene and polycarbonate, cellulose, unqualified to this.These base materials can have any shape according to required purposes, for example, and forms such as microplate and pearl, film, chromatostrip, sheet, pipe, pin or groove shape body.
About the implication of the term " electrolytic thin-membrane " that uses in this instructions, in general, term " electrolyte " uses water miscible material, but when making the electrolytic thin-membrane that can use in the present invention, not necessarily must use water-soluble material.For example, even charged corpuscle etc. are insoluble, oily material, as long as use with the state that is dispersed in the organic solvent, the material that also can be used as electrolytic thin-membrane of the present invention uses.In addition, the material of electrolytic thin-membrane of the present invention need not be polymkeric substance (macromolecular material), also can use inorganic material beyond the macromolecular material etc., but aspect filming and reduction manufacturing cost, more preferably macromolecular material.
Be suitable as electrolytic polymer, this macromolecular material can preferably use electronegative electrolytic polymer, as polyacrylic acid, polymethylacrylic acid, polystyrolsulfon acid, poly-to water-soluble anionic polymers such as benzene phenylene (-), polythiophene-3-acetate, polyamide, as the positively charged electrolytic polymer, can use polyarylamine hydrochloride, poly dimethyl diaryl ammonium chloride, polypyrrole, polyaniline, poly-to water-soluble cationic polymers such as benzene phenylene (+), polyparaphenylene vinylene, poly-ethyl imines, unqualified to this.
Of the present invention be arranged at immobilization can play with the electrolytic thin-membrane of the substrate surface of carrier specificity on the surface at this carrier, effectively and repeatability well in conjunction with the effect of the adsorption film of varied material, it can be formed by thin film, and perhaps the laminated film that is made of the multi-layer thin rete forms.As laminated film, the occasion forming above-mentioned electrolytic thin-membrane can form this electrolytic thin-membrane with the 1st positively charged charged (electrolyte) film and the 2nd electronegative charged membrane combination in any and lamination.In general, consider repelling each other between the like charges, preferred the 1st charged membrane and the 2nd charged membrane alternatively laminated.In addition, positively charged multiple charged membrane and electronegative multiple charged membrane also can be arbitrarily or the electric charge alternatively laminated.
No matter form the film of electronegative still positively charged, in the occasion that forms the electrolytic thin-membrane that constitutes by one deck, can be in the aqueous solution of above-mentioned anionic polymer or cationic polymer aqueous solution the surface of impregnated carrier base material.
In addition, forming the occasion of electrolytic thin-membrane as laminated film, can be arbitrarily or the electrolytic thin-membrane and the lamination of electronegative electrolytic polymer film of alternate combinations and positively charged, use G.Decker, ThinSolid Films, 210/211,831 (1992), D.S.Yoo, Macromolecules, 31,4309 (1998), He Keyan is arranged, chemical industrie, the 52nd volume, No. 7, the method for putting down in writing in the 853-856 page or leaf can form a kind of alternately adsorbed layer press mold as the lamination of the electrolytic thin-membrane of electronegative thin polymer film and positively charged (absorption) film.In general, can carry out the operation of alternating impregnating carrier in electrolytic thin-membrane (kation) aqueous solution of electronegative thin polymer film (negative ion) aqueous solution and positively charged.
In addition, except that dipping, can also use injection tube, suction pipe, dispenser, bristle brush, inkjet nozzle, impression or printing etc., the optional position on carrier, drip coating spraying impression polymer solution, at random form electrolytic thin-membrane of the present invention at carrier surface.And, also can by use the atomic force microscope probe dip a nanometer lithography (DPN, Dip-PenNanolithography), in extremely small zone coating.
There is no particular limitation to the thickness of the electrolytic thin-membrane that forms, preferred 1 layer thickness is 0.1～50nm, several 1～1000 layer of lamination, thickness 0.001～50 μ m, more preferably 1 layer thickness is 0.1～50nm, several 1～500 layer of lamination, thickness 0.001～25 μ m, preferred especially 1 layer thickness is 0.1～50nm, several 1～100 layer of lamination, thickness 0.001～5 μ m.Preferred 4～60 ℃ of temperature when film forms, more preferably 10～45 ℃, preferred especially 15～37 ℃.In addition, there is no particular limitation to time of flooding in anionic polymer aqueous solution or cationic polymer aqueous solution, and preferred 1～60 minute, more preferably 3～30 minutes, preferred especially 3～15 minutes.
When above-mentioned alternating layer press mold forms, forms 1 layer after, can form another layer rapidly, also can after several days or several hours, form another layer again.Film forms the general water flush vehicle of washing procedure of operation and finishes washing.As long as formation does not have obstacle to film, and is unqualified to the purity of water.In addition, washing not necessarily must be a water also with liquid, also can use the solvent that can dissolve polyelectrolyte.
In addition, as mentioned above, general terms " electrolyte " is meant water-soluble material used, and making can be used for film of the present invention the time, not necessarily must use the material of aqueous solution.For example, also can use charged corpuscle insoluble material and oily materials such as (for example, the ferrite particulate), as long as use with the state that is dispersed in organic solvent or the water, just can be as the material of dielectric film of the present invention.The material of dielectric film of the present invention also not necessarily must be a polymkeric substance (macromolecular material).For example, also can use the metal complex monomer, perhaps use inorganic oxide as anionic membrane as cationic membrane.
Immobilization after film forms can be preserved at normal temperatures with carrier, has the stability more than 1 year usually.
Afterwards, immobilization of the present invention with carrier on, can the various materials of immobilization, for example combine or have the material of compatibility with them by the material histocyte thalli virus of biosome origin or with them, especially protein, glycoprotein, peptide, glycopeptide, polysaccharide, nucleic acid, lipid, glycolipid matter, vitamins, steroids, steroids such as catecholamine and thyroxine, prostanoid, GABA, acetylcholine, various transmitter substances such as serotonin and dopamine, the material by the biosome origin of microbiotic representative, cell or the material that combines with them or have material of compatibility etc. with them, during immobilization, use to well known to a person skilled in the art that any method all can.Like this, the solid phase of having fixed various materials on carrier is preferred for utilizing in the hybridization, receptor assay, biology sensor etc. of various antigen-antibody reactions, the nucleic acid of this solid phase.
As mentioned above,, can make immobilization carrier, the solid phase with excellent specific property can be provided with very shirtsleeve operation operation with excellent ability to cure and stability according to the present invention.
According to immobilization carrier of the present invention, at the substrate surface coating electrolytic thin-membrane of immobilization with carrier, can be effectively and with good repeatability at the fixing desired substance of the carrier surface of this coating, can provide immobilization with carrier with utilize the solid phase of this carrier with excellent specific property.
Embodiment
Illustrate in greater detail the present invention below by embodiment, the present invention is not subjected to the qualification of following embodiment certainly.
(embodiment 1)
In polyacrylic acid aqueous solution (concentration 10
-2M) in, under 25 ℃, the microplate in 96 holes of dipping washed with water after 15 minutes.At this moment, the pH of polyacrylic acid aqueous solution remains on 3.5.On the other hand, be prepared as follows solution: in 2 (N-morpholinyl) ethane sulfonic acid (MES) damping fluids (pH6) of 0.1M, the few deoxyribosylthymine (20mer) that adding makes 5 '-terminal amino groupization further adds 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to 25mM to 1.3mM.The solution 75 μ L that add preparation in each hole of above-mentioned plate 60 ℃ of reactions 6 hours, are fixed on internal surface of hole with few deoxyribosylthymine.After water washes unreacted few deoxyribosylthymine, 10mM three (hydroxyl) aminomethane hydrochloride-1mM ethylenediamine-N that adds the luciferase assay reagent OliGreen that contains 0.5vol%, N, N ', N '-tetraacethyl (TE) damping fluid (pH8.0) 75 μ L carry out fluorometric assay.Perhaps, do not have also to add in the immobilized hole luciferase assay reagent in few deoxyribosylthymine, measure fluorescence intensity, difference is as blank.
The test findings of n=8 is as shown in table 1, and in the gained microplate, the fluorescence intensity of each micropore is all strong as can be seen, the most of combinations of few deoxyribosylthymine.
(Comparative Examples 1)
The synthetic quartz system of use Cooper-Hewitt lamp is to the microplate irradiation 200mJ/cm in 96 holes
2Ultraviolet ray.Use this microplate, the same with embodiment 1, few deoxyribosylthymine is fixed on internal surface of hole, below carry out the operation the same with embodiment 1.
The test findings of n=8 is as shown in table 1, and in the gained microplate, a little less than the fluorescence intensity ratio embodiment 1, repeatability is also poor.
Table 1
| The sequence number in hole | Fluorescence intensity | |
| Embodiment 1 | Comparative Examples 1 | |
| ????1 | ????H | ????L |
| ????2 | ????H | ????L |
| ????3 | ????H | ????M |
| ????4 | ????H | ????L |
| ????5 | ????H | ????L |
| ????6 | ????H | ????M |
| ????7 | ????H | ????M |
| ????8 | ????H | ????L |
(fluorescence intensity) H:＞5000, M:2000～5000, L:＜2000
(embodiment 2)
In polyacrylic acid aqueous solution (concentration 10
-2M) in, under 25 ℃, the microplate in 96 holes of dipping washed with water, then in polyarylamine aqueous solution (concentration 10 after 15 minutes
-2M) in, under 25 ℃, the microplate after 15 minutes in 96 holes of dipping washes with water, with its alternate repetition 10 times, forms the alternating layer press mold.At this moment, the pH of two electrolyte aqueous solutions remains on 3.5.In 100mM sodium phosphate buffer (pH7.0), dissolve acarid antigen protein (Der f II, biochemical industry system) and, in the hole of the flat board that forms the alternating layer press mold, add 100 μ L, adsorb a night at 4 ℃ so that it reaches the concentration of 5 μ g/mL.Behind PBS washing hole 3 times, add 1% bovine serum albumin solution (the Diluent/Blocking Solution Concenrate10 of usefulness distilled water diluting KPL society system is doubly) of 400 μ L, 25 ℃ of reactions 1 hour, seal unreacted solid phase.Behind PBS washing hole 3 times, add the serum 100 μ L of the acarid autopath with the special IgE antibody of acarid in the hole, 25 ℃ of reactions 2 hours.Behind PBS washing hole 3 times, add the anti-people's of the peroxidase sign that is modulated into 1 μ g/mL IgE antibody (KPL society system) 100 μ L, 25 ℃ of reactions 1 hour.Behind PBS washing hole 5 times, add TMB (KPL society system 3,3 ', 5,5 '-tetramethyl benzidine) 100 μ L, reacted 3 hours, add 100 μ L 1M phosphoric acid then, cessation reaction.Use microplate, measure the absorbance of 450nm.As negative control, the serum that adds healthy people replaces acarid autopath's serum, also carries out same operation.Obtain standard deviation (SD) from 5 mensuration of negative control, further the value of the mean value addition that numerical value and 5 times of its 2 times of changes are measured is as MEAN+2SD.On the other hand, measure from 5 times of acarid autopath serum and to obtain standard deviation (SD), further will deduct value that the numerical value of its 2 times of changes obtains as MEAN-2SD with mean value.The value that deducts MEAN+2SD with MEAN-2SD be 0 or negative value as negative (-), on the occasion of as the positive (+).
It the results are shown in the table 2.As can be seen from Table 2, can judge all acarid autopaths positive (+).
(Comparative Examples 2)
With the Cooper-Hewitt lamp of synthetic quartz system, to the microplate irradiation 200mJ/cm in 96 holes
2Ultraviolet ray.Use this microplate, the same with embodiment 2, carry out the immobilization of acarid antigen protein, implement operation and the calculating the same below with embodiment 2.As shown in table 2, can not judge in the gained microplate all acarid autopaths positive (+).
Table 2
| The serum name | Embodiment 2 | Comparative Examples 2 | |
| The acarid autopath | Serum 1 | Positive (+) | Positive (+) |
| Serum 2 | Positive (+) | Negative (-) | |
| Serum 3 | Positive (+) | Positive (+) | |
| Serum 4 | Positive (+) | Positive (+) | |
| Serum 5 | Positive (+) | Negative (-) | |
| Serum 6 | Positive (+) | Negative (-) | |
| Serum 7 | Positive (+) | Negative (-) | |
| Serum 8 | Positive (+) | Negative (-) | |
(embodiment 3)
In the microplate in 96 holes, inject polyacrylic acid aqueous solution (concentration 10
-2M pH5.0), placed 3 minutes at 25 ℃, washed with water, injected polyacrylic acid aqueous solution (concentration 10 then
-2M pH2.5), placed 3 minutes at 25 ℃, washed with water.With its alternate repetition 2.5 times, form the alternating layer press mold.100mM sodium phosphate buffer (PBS) (pH7.0) in dissolving make concentration reach 1 μ g/ μ L from the aldehyde lyase of rabbit origin, in the hole of the flat board that forms the alternating layer press mold, add 100 μ L, adsorbed for 1 night at 4 ℃.Behind PBS washing hole 3 times, add 1% bovine serum albumin solution (the Diluent/Blocking Solution Concenrate10 of usefulness distilled water diluting KPL society system is doubly), 360 μ L, 25 ℃ of absorption 1 hour, seal.Remove albumin solution, add the PBS solution 100 μ L of the aldehyde lyase specific antibody of peroxidase sign in the hole, 25 ℃ of reactions 2 hours.Behind PBS washing hole 4 times, add 100 μ L TMB (KPL society system 3,3 ', 5,5 '-tetramethyl benzidine), and reacted 15 minutes, add 100 μ L 1M phosphoric acid cessation reactions.Measure the absorbance of 450nm with microplate.The test findings of n=8 is as shown in table 3, in the hole that forms the alternating layer press mold, and the absorbance height.
(Comparative Examples 3)
The same with embodiment 3, make the microplate in 96 holes that do not form the alternating layer press mold adsorb aldehyde lyase, after the sealing, add the aldehyde lyase specific antibody of peroxidase sign, by the TMB reaction, measure absorbance.As shown in table 3, absorbance than embodiment 3 a little less than.
Table 3
| The sequence number in hole | Embodiment 3 | Comparative Examples 3 |
| ????1 | ????H | ????L |
| ????2 | ????H | ????L |
| ????3 | ????H | ????M |
| ????4 | ????H | ????L |
| ????5 | ????H | ????M |
| ????6 | ????H | ????L |
| ????7 | ????H | ????M |
| ????8 | ????H | ????M |
(absorbance) H:＞1.4, M:1.0-1.4, L:＜1.0
(embodiment 4)
In the microplate in 96 holes, inject polyacrylic acid aqueous solution (concentration 10
-2M pH5.0), placed 3 minutes at 25 ℃, washed with water, injected polyacrylic acid aqueous solution (concentration 10 then
-2M pH2.5), placed 3 minutes at 25 ℃, washed with water.With its alternate repetition 4.5 times, form the alternating layer press mold.100mM sodium phosphate buffer (PBS) (pH7.0) in dissolving to make concentration by the lysozyme of albumen origin be 1ng/ μ L, in the hole of the plate that forms the alternating layer press mold, add 100 μ L, adsorbed for 1 night at 4 ℃.Behind PBS washing hole 3 times, add 1% bovine serum albumin solution (10 times of the Diluent/Blocking Solution Concenrate of usefulness distilled water diluting KPL society system), 360 μ L, 25 ℃ of absorption 1 hour, seal.Remove albumin solution, add the PBS solution 100 μ L of the lysozyme specific antibody of peroxidase sign in the hole, 25 ℃ of reactions 2 hours.Behind PBS washing hole 4 times, add 100 μ L TMB (KPL society system 3,3 ', 5,5 '-tetramethyl benzidine), and reacted 15 minutes, add 100 μ L 1M phosphoric acid cessation reactions.Measure the absorbance of 450nm with microplate.The test findings of n=8 is as shown in table 4, in the hole that forms the alternating layer press mold, and the absorbance height.
(Comparative Examples 4)
The same with embodiment 4, make the microplate in 96 holes that do not form the alternating layer press mold adsorb lysozyme, after the sealing, add the lysozyme specific antibody of peroxidase sign, by the TMB reaction, measure absorbance.As shown in table 4, absorbance than embodiment 4 a little less than.
Table 4
| The sequence number in hole | Embodiment 3 | Comparative Examples 3 |
| ????1 | ????H | ????L |
| ????2 | ????H | ????L |
| ????3 | ????H | ????L |
| ????4 | ????H | ????L |
| ????5 | ????H | ????L |
| ????6 | ????H | ????L |
| ????7 | ????H | ????M |
| ????8 | ????H | ????L |
(absorbance) H:＞1.2, M:0.8-1.2, L:＜0.8
Claims (8)
1. an immobilization carrier is characterized in that, on the surface of carrier electrolytic thin-membrane is set.
2. the described immobilization carrier of claim 1, described electrolytic thin-membrane is formed by macromolecular material.
3. the described immobilization carrier of claim 2, described electrolytic thin-membrane is made of polyanion film or any of polycation film.
4. the described immobilization carrier of claim 2, described electrolytic thin-membrane is made of polyanion film and polycation film alternatively laminated.
5. each described immobilization carrier of claim 1～4 is used for fixing material that combines with the detection thing or the material that has compatibility with the detection thing.
6. each described immobilization carrier of claim 1～5 is used for fixing protein, glycoprotein, peptide, glycopeptide, polysaccharide, nucleic acid, lipid, glycolipid matter etc. and has the material of compatibility by material, cell or the material that combines with them of biosome origin or with them.
7. solid phase, each described immobilization of claim 1～4 with carrier on, fixing with detect material that thing combines or the material that has compatibility with the detection thing.
8. solid phase, each described immobilization of claim 1～4 with carrier on, fixing protein, glycoprotein, peptide, glycopeptide, polysaccharide, nucleic acid, lipid, glycolipid matter etc. have the material of compatibility by material, cell or the material that combines with them of biosome origin or with them.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002267636 | 2002-09-13 | ||
| JP267636/2002 | 2002-09-13 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1682113A true CN1682113A (en) | 2005-10-12 |
| CN100339712C CN100339712C (en) | 2007-09-26 |
Family
ID=31986711
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB038216086A Expired - Fee Related CN100339712C (en) | 2002-09-13 | 2003-09-12 | Fixation carrier and solid phase |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US20060051762A1 (en) |
| EP (1) | EP1548439A4 (en) |
| JP (1) | JPWO2004025300A1 (en) |
| CN (1) | CN100339712C (en) |
| AU (1) | AU2003261568B2 (en) |
| WO (1) | WO2004025300A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106573220A (en) * | 2014-07-24 | 2017-04-19 | 凸版印刷株式会社 | Lipid membrane structure, lipid-membrane-structure-immobilization carrier, and method for fusing cells |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012117895A (en) * | 2010-11-30 | 2012-06-21 | Hitachi Chem Co Ltd | Metal detection sensor, method for adsorbing metal and method for determining metal concentration |
| JP6420055B2 (en) * | 2013-03-29 | 2018-11-07 | 積水メディカル株式会社 | Colored particles for immunochromatography and diagnostic immunochromatography reagent using the same |
| JP6540032B2 (en) * | 2015-01-19 | 2019-07-10 | 日立化成株式会社 | Separation material |
| CN111377533B (en) * | 2020-04-21 | 2020-12-01 | 山东高速环保科技有限公司 | Sewage treatment microbial carrier and preparation method thereof |
Family Cites Families (15)
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| JPH0622632B2 (en) * | 1987-09-08 | 1994-03-30 | 鐘淵化学工業株式会社 | Adsorbent and removal device |
| CA1327963C (en) * | 1987-09-08 | 1994-03-22 | Ryuichi Yokohari | Autoantibody adsorbent and apparatus for removing autoantibodies using the same |
| JP2614905B2 (en) * | 1988-09-22 | 1997-05-28 | 帝人株式会社 | Immunosensor |
| JPH0750111B2 (en) * | 1988-12-22 | 1995-05-31 | 積水化学工業株式会社 | Carrier for immobilizing hydrophobic substance and method for immobilizing hydrophobic substance using the same |
| DE4208645A1 (en) * | 1992-03-18 | 1993-09-23 | Bayer Ag | OPTICAL SOLID PHASE BIOSENSOR BASED ON FLUORESCENT COLOR-MARGINED POLYIONIC LAYERS |
| US5629213A (en) * | 1995-03-03 | 1997-05-13 | Kornguth; Steven E. | Analytical biosensor |
| DE19530078A1 (en) * | 1995-08-16 | 1997-02-20 | Bayer Ag | Optical solid phase biosensor based on streptavidin and biotin |
| DE69803129T2 (en) * | 1997-02-14 | 2002-08-29 | Nippon Catalytic Chem Ind | Immobilized biocatalyst |
| DE19810965A1 (en) * | 1998-03-13 | 1999-09-16 | Aventis Res & Tech Gmbh & Co | Nanoparticles comprising polyelectrolyte complex of polycation, polyanion and biologically active agent, especially useful for controlled drug release on oral administration |
| EP1162459A1 (en) * | 2000-06-07 | 2001-12-12 | Corning Incorporated | Rough charged solid phase for attachment of biomolecules |
| WO2002054052A1 (en) * | 2001-01-08 | 2002-07-11 | Leonard Fish | Diagnostic instruments and methods for detecting analytes |
| JP2002350447A (en) * | 2001-05-24 | 2002-12-04 | Wako Pure Chem Ind Ltd | Physiological active material fixing carrier, method of manufacturing the same fixing physiological active material, method of analyzing object component in sample and kit for analyzing object component in sample |
| US6689478B2 (en) * | 2001-06-21 | 2004-02-10 | Corning Incorporated | Polyanion/polycation multilayer film for DNA immobilization |
| US7112361B2 (en) * | 2001-10-25 | 2006-09-26 | Massachusetts Institute Of Technology | Methods of making decomposable thin films of polyelectrolytes and uses thereof |
| WO2003035278A1 (en) * | 2001-10-25 | 2003-05-01 | Massachusetts Institute Of Technology | Method of depositing polyelectrolyte multilayers and articles coated thereby |
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2003
- 2003-09-12 CN CNB038216086A patent/CN100339712C/en not_active Expired - Fee Related
- 2003-09-12 WO PCT/JP2003/011710 patent/WO2004025300A1/en active Application Filing
- 2003-09-12 JP JP2004535962A patent/JPWO2004025300A1/en active Pending
- 2003-09-12 AU AU2003261568A patent/AU2003261568B2/en not_active Ceased
- 2003-09-12 EP EP03795428A patent/EP1548439A4/en not_active Withdrawn
- 2003-09-12 US US10/527,805 patent/US20060051762A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106573220A (en) * | 2014-07-24 | 2017-04-19 | 凸版印刷株式会社 | Lipid membrane structure, lipid-membrane-structure-immobilization carrier, and method for fusing cells |
| CN106573220B (en) * | 2014-07-24 | 2019-08-16 | 凸版印刷株式会社 | The fusion method of lipid membrane structure body, lipid membrane structure body fixation support and cell space |
Also Published As
| Publication number | Publication date |
|---|---|
| US20060051762A1 (en) | 2006-03-09 |
| EP1548439A1 (en) | 2005-06-29 |
| WO2004025300A1 (en) | 2004-03-25 |
| AU2003261568B2 (en) | 2006-12-07 |
| EP1548439A4 (en) | 2008-04-23 |
| JPWO2004025300A1 (en) | 2006-01-12 |
| AU2003261568A1 (en) | 2004-04-30 |
| CN100339712C (en) | 2007-09-26 |
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